Poly(ethylcyanoacrylate) (PECA) nanospheres have been employed as biodegradable polymeric carriers for oral (po) delivery of insulin. The main goal of this investigation was to screen various absorption enhancers, which were used to protect insulin-loaded PECA, by following their in vivo performance after oral administrations to streptozotocin-induced diabetic rats. The nanospheres were prepared by polymerization in a continuous aqueous phase at pH 2.5 and in the presence of Pluronic 68 (0.5%). This polymerization technique was able to hold 85 +/- 7.5% of insulin added, 30 min after initiation of polymerization. The drug loading was evaluated by HPLC. Insulin absorption after oral administration was evaluated by its hypoglycemic effect. The addition of protease inhibitor to insulin-loaded PECA nanospheres significantly reduced the blood glucose level after po administrations. Capric acid (0.5%) showed the maximum reduction in blood glucose level (bG max = 34.4 +/- 4.1 mg%), while cholic acid (0.5%) showed the fastest reduction in blood glucose level (Tmax, G = 6.0 +/- 0.2 h) among the tested oral absorption enhancers. Insulin associated with PECA nanospheres retained its biological activity up to 12 days in 50% of the rats in the presence of glycyrrhizic acid (1%). The per cent pharmacological availabilities (PA%) were in the order of capric acid (0.5%) > glycyrrhizic acid (1%) > deoxycholic acid (0.5%) > hydroxypropyl-beta-cyclodextrin (HPbetaCD) (1%) > cholic acid (0.5%). There was no significant difference in the PA% between capric acid (0.5%), glycyrrhizic acid (1%) and deoxycholic acid (0.5%). Although sodium cholate (0.5%) showed the lowest increase in PA% (3.1 +/- 3.11%), its Tmax G was the shortest (6 +/- 0.2 h) among the tested absorption enhancers. The reduction in blood glucose levels was maintained < 200 mg/dL in the order of glycyrrhizic acid (166 +/- 56 h) > capric acid (72 h) >; deoxycholic acid (66 +/- 6 h) > no enhancer (64 +/- 16 h) > HPbetaCI) (31 +/- 17.5) > cholic acid (22 +/- 5.7 h) > aprotenin (20 +/- 3.5 h). Therefore, capric acid and glycyrrhizic acid could be successfully used as oral absorption enhancers.